Signal transduction cascades maintain control over important cellular processes such as cell growth and differentiation by orchestrating protein phosphorylation and dephosphorylation. Specific control of these processes in vivo and in vitro can be achieved with peptide analogues that mimic the binding properties of phosphoproteins. We present here the solid-phase synthesis of two novel classes of phosphopeptide mimetics, O-boranophosphopeptides and O-dithiophosphopeptides, derivatized on tyrosine, serine, and threonine. The use of H-phosphonate and H-phosphonothioate monoesters containing the base labile 9-fluorenemethyl protecting group was key to the synthesis of both phosphopeptide mimetics. O-Boranophosphopeptides were synthesized by condensing O-(9-fluorenemethyl)-H-phosphonate to the peptide hydroxylic component (tyr, ser, or thr) followed by oxidation with borane complexes. Similarly, the synthesis of O-dithiophosphopeptides used the O-(9-fluorenemethyl)-H-phosphonothioate synthon and oxidation with elemental sulfur. Base elimination of the Fmol protecting group and cleavage from the solid support with concentrated ammonium hydroxide afforded the boranophosphopeptide and dithiophosphopeptide target compounds. Ac-YIIPLPG-NH2, having either dithiophosphoryl tyrosine or boranophosphoryltyrosine but no sequence specificity for Yersinia protein tyrosine phosphatase (PTP), was found to competitively inhibit this enzyme with KI values of 430 +/- 50 and 670 +/- 50 microM, respectively. In addition, both phosphopeptide analogues were resistant toward Yersinia PTP enzymatic hydrolysis. Under conditions (pH 8.0) where the phosphopeptide was rapidly dephosphorylated, the boranophosphopeptide hydrolyzed slowly (t1/2 = 15 h) and the dithiophosphopeptide was completely stable over 24 h.